The invention relates to a laser microdissection method with the aid of which a dissectate is cut out along a closed cutting line from a biological specimen, which is mounted on a planar carrier, by means of laser pulses of a laser.
Furthermore, the invention relates to a device for laser microdissection that comprises a microscope having at least one objective defining an optical axis. Furthermore, a pulsed laser is provided that emits a laser beam that is directed along the optical axis onto a specimen via the objective and describes a closed cutting line.
In the field of biology and medicine, microdissection denotes a method with which a small piece, a so-called dissectate, is cut out of a generally flat specimen (for example cells, cell cultures or a tissue section) with the aid of a focused laser beam. The biological specimen is mounted for laser cutting on a planar carrier, for example a glass specimen slide or a polymer film. The dissectate is available after the cut for further biological or medical (for example histological) examinations.
Such a method for a laser microdissection is described in the article entitled “Cell surgery by laser microdissection: a preparative method” by G. Isenberg, W. Bilser, W. Meier-Ruge, E. Remy, Journal of Microscopy, vol. 107, May 1976, pages 19-24. A biological specimen is mounted there on the underside of a specimen slide. What is meant by biological specimen is cell cultures that have been attracted on a specimen slide. In order to prevent a permanent adhesion of these cells on the substrate, use is made of silicone-coated specimen slides that effect a reduction in the adhesion between specimen and specimen slide. The specimen slide lies in an erect microscope into which a pulsed He—Ne laser is coupled. The laser beam is focused onto the biological specimen. A specimen field of interest, the dissectate, is cut out along a closed cutting line by juxtaposing cut holes produced by the laser pulses with the aid of the focused laser beam. The cutting is based in this case on the known principle of laser ablation, that is to say the individual laser pulses produce on the cutting line a plasma that “vaporizes” the specimen material. In this case, the last laser pulse separates the dissectate from the surrounding biological specimen and in so doing it also effects the required loosening of the dissectate from the specimen slide. The dissectate then falls down under the action of gravity, and is captured in a collecting vessel and fed to further examinations.
DE 100 43 506 C1 describes a further development of this method. In this case, the specimens to be examined and from which specimen fields of interest are to be cut out are prepared on very thin plastic films. The thickness of these plastic films is of the order of magnitude of 1-2 μm. PET films and PEN films come into question as material. The specimen is loaded into a microscope into which a pulsed laser is coupled. A method for laser microdissection is described in which the cutting line is not completely closed toward the end of the cut, but a narrow and at the same time stable web remains at the end. This prevents the film with the specimen field of interest from being swung out and twisted outside the focal plane. Before the web is severed, the aperture of the laser beam is enlarged by means of a diaphragm without varying the observation aperture of the microscope. The cutting width of the laser beam is enlarged by the enlarged laser aperture. At the same time, the position of the focus of the laser beam is kept without variation at the same position relative to the specimen. The residual web is then severed with the expanded laser aperture with the aid of a last, focused, cutting laser pulse. At the termination of the cut, the specimen falls down under the action of gravity and is collected in a collecting vessel. However, it has emerged overall that it is complicated in terms of equipment and time-consuming to stop the cutting line before the last laser pulse and to switch over the diaphragm for the laser aperture before the cutting line is terminated with the last laser pulse. Again, it proves not to be quite so simple for the user to fix a suitable residual web and to assign a fitting laser aperture, and so the dissectates are sometimes not entirely freely prepared and the cut has to be repeated.
Moreover, it has been observed in the case of both methods that when the equipment setting (optics, laser parameters, focal position etc.) is not varied, dissectates sequentially cut out drift away laterally to a different extent when they fall into the collecting apparatus. This collecting apparatus can be, for example, a specimen tube, usually referred to in the market as a PCR tube. The consequence is then that the dissectates adhere laterally to the inner wall of the PCR tube instead of falling to the bottom of the tube. It is then difficult for them to be inspected, and this constitutes for the user, for example a pathologist, a substantial working step before the further processing of the cutout dissectates.
Consequently, German patent application DE 103 46 458 proposes a method for laser microdissection of a specimen field of interest of a specimen in which the laser pulses of a pulsed laser beam are likewise focused on the specimen, and in the case of which the mass ablated at the last laser pulse completing the cut is adapted to the cutting width of the last cutting laser pulse and optimized so as to maximize the energy transferred from the plasma on to the dissectate.
However, the stopping of the cutting line before the last laser pulse is felt to be time-consuming by the user here too.
U.S. Pat. No. 6,773,903 likewise discloses a method for microdissection in which selected fields of a biological specimen are cut out. The specimen mounted on the specimen slide lies on a stage movable in the x-y coordinate plane. A laser beam is coupled into the microscope and the x-y stage is appropriately moved such that this laser beam describes an appropriately closed cutting line about the specimen field of interest. Consequently, the biological material of interest is separated from the biological specimen. The control of the x-y stage is, however, mechanically complex and not so accurate as if the laser beam were controlled appropriately in the x-y plane in order to separate the biological material from the remainder of the specimen.
It is therefore an object of the invention to specify a method for laser microdissection that permits the dissectate to be cut out in a more comfortable and speedier fashion accompanied by further improved cutting results even in the case of difficult specimen preparation.
This object is achieved by a laser microdissection method described herein.
A further object of the invention is to specify a device for laser microdissection with which the user can obtain the desired dissectates precisely, quickly and reliably. In this case, obtaining the dissectates is independent of the respective specimen preparation.
This object is achieved by a device for laser microdissection described herein.